1. Field of the Invention
This invention relates to in vitro fertilization (IVF) and to cloning of animals, and to methods for improving the efficiency of IVF and cloning as well as increasing fertilization rates in IVF
2. Description of the Related Art
Reproductive wastage is a universal characteristic of biology, with all forms of life devoting enormous energies toward production of germ cells far in excess of the number that eventually develop into a new adult capable of repeating the life cycle. Ovaries of mammals, including women and domestic animal species, contain hundreds of thousands of germ cells at birth, the majority of which never are ovulated, being lost by atretic processes at various stages of follicle development, before puberty or in adult life.
Understanding the basic mechanisms that control the natural selection of the relatively few oocytes that are ovulated, can provide the key to tapping this enormous genetic resource. Applied to women, this knowledge will produce new insights into causes of ovarian dysfunction, and can possibly lead to improved procedures for the diagnosis of infertility, and reduce the risk of high multiple gestations generated by empiric infertility therapies.
Multiple gestations are increasing at an alarming rate due to the growing use of infertility treatments. Presently, 77% of triplets result from assisted reproduction technologies (ART's). Between 1980 and 1994, 10% of the 37,514 triplets, quadruplets, and other-higher multiplies died in their first year, according to the National Center for Health Statistics (Belluck, 1998). Multiple pregnancies suffer a five fold higher stillbirth rate than singleton pregnancies. Of those that survive, 92% are born prematurely and below normal birth weight, which can lead to health and developmental problems. Triplets are twice as likely to develop blindness, mental retardation or seizure disorders as singletons (Belluck, 1998). The rate of cerebral palsy in multiple gestation is 12 times that of singleton pregnancies (Crether, 1993). In a study of 13,206 pregnancies at a Boston hospital, the average cost for postpartum care of triplets was $109,000 (Callahan, 1994).
Theoretically, culture to the blastocyst stage of development would enable embryos to self-select. This technique has proved valuable to selected patients who produce large numbers of healthy-appearing embryos (Meldrum. 1998), which are likely to tolerate the additional days in culture. However, with the increase in the average age of women undergoing IVF, who exhibit less robust responses to controlled ovarian stimulation, blastocyst transfer has less clear value. In this patient group, the majority of embryos do not survive five days in culture, and the embryos which do survive may not exhibit superior implantation rates compared to embryos transferred on the conventional third day. Moreover, even cases of blastocyst transfer present the dilemma of which blastocysts to transfer.
Most clinical embryo viability scoring systems currently used in IVF laboratories focus on embryo morphology. However, because the oocyte serves as the “stem cell” for the embryo, and because more than 80% of aneuploidies that appear in preimplantation embryos originate in the oocyte spindle structure, the evaluation of oocyte structure and determination of fertilization and developmental potential is important, and examination of an important structure in oocytes, the meiotic spindle, is key.
Evaluation of oocyte quality has been difficult in humans. Attempts to estimate oocyte development potential demonstrate a number of morphologic features associated with poor developmental potential, such as darkness, granularity, vacuoles, fragmentation and irregularity (Bolton, 1989, Weimer, 1993, Riley, 1991, Fleming, 1982), but in fact, such standard imaging techniques do not provide a sensitive method of diagnosing oocyte dysfunction. Moreover, the pathobiological basis of these morphological markers is unclear.
IVF offers the opportunity to study the role of the meiotic spindle in human oocyte developmental potential, because oocytes are ovulated at the MII stage of development, when the chromosomes are poised on the metaphase plate, tethered by microtubules that are inherently unstable, and relative to other structures in the oocyte, highly birefringent. In patients who undergo immature oocyte retrieval and IVM, the MI spindle also is available for analysis. Unfortunately, the imaging methods currently used in the IVE laboratory, e.g., Hoffmnan, Nomarski or bright field microscopy, cannot image clearly the meiotic spindle.
A previous study (Battaglia, 1996) compared spindles of oocytes from two groups of women, aged 20 to 25, and aged 40 to 45 years using immunofluorescence and high-resolution, confocal microscopy, and found that meiotic spindles from older women exhibited significantly more abnormalities in chromosome placement and structure. In the older group, 79% of oocytes from the older group exhibited abnormal spindle structure, including abnormal tubulin placement and displacement of one or more chromosomes from the metaphase plate. In the younger group, only 17% exhibited such abnormalities. Spindles in the younger group appeared well ordered, and held chromosomes aligned on the metaphase plate. This data suggests that the architecture of the meiotic spindle is altered in older women, possibly explaining their higher prevalence of aneuploidy.
While intriguing, these results originated from experiments that destroyed the oocytes by fixing and staining them and illuminating them with intense, high frequency light. Moreover, because it employed invasive imaging, it could not link spindle architecture to developmental outcome.
Oocytes, like most living cells, are almost entirely translucent when viewed with a standard optical microscope, making it necessary to employ methods for creating and enhancing contrast in order to discern cellular components. Nomarksi (also called differential interference contrast or DIC), Hoffman, phase contrast, and traditional polarized light techniques use optical interference effects to create contrast, while other imaging methods require marking specific cellular components with exogenous, absorptive colored stains or fluorescent labels. While producing high spatial resolution, these latter methods either kill the cell or affect its function, and therefore, provide limited value to clinical and/or developmental studies.
Birefringence is an optical property that derives from the molecular order found in such macromolecules as membranes, microtubules, microfilaments, and other cytoskeletal components. Polarized light microscopy has the unique potential to visualize and measure birefringent structures, such as spindles dynamically and non-destructively in living cells. However, the low sensitivity of conventional polarized light microscopes makes them marginally suitable for application to mammalian experimental and clinical embryology. (Oldenbourg. 1995, Oldenbourg. 1996. Oldenbourg 1997). In polarized light microscopy, birefringence is measured as retardance which arises when the optical paths between two orthogonal, polarized light beams are differentially slowed as they pass through highly ordered molecules, such as microtubules within the specimen. Birefringent objects, such as spindles, present differences in the paths encountered by polarized light beams as they pass through the object. Compared to non-biological materials, the birefringence of biological samples is weak, only a few nanometers, so the relatively low level of birefringence in biological specimens requires the use of manually-adjusted compensators and rotating stages, a complicated procedure which is prohibitively slow for clinical applications. Moreover, the quantification of retardance levels, necessary to compare spindles, is complicated with conventional polarized light microscopes because the signal achieved originates from both the inherent birefringence in the specimen and the setting of the manually adjusted compensation and analyzers.